CN111217935A - Micromolecule citrus pectin and preparation method thereof - Google Patents
Micromolecule citrus pectin and preparation method thereof Download PDFInfo
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- CN111217935A CN111217935A CN201811411764.4A CN201811411764A CN111217935A CN 111217935 A CN111217935 A CN 111217935A CN 201811411764 A CN201811411764 A CN 201811411764A CN 111217935 A CN111217935 A CN 111217935A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
- C08B37/0048—Processes of extraction from organic materials
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Abstract
The invention discloses a preparation method of micromolecule citrus pectin, aiming at providing a preparation method of micromolecule citrus pectin, which has simple operation, single molecular weight distribution and easy industrial production; the technical key points are as follows: (1) dissolving pectin in deionized water to form a solution; (2) centrifuging the pectin solution to remove insoluble impurities; (3) separating by microfiltration of pectin solution; (4) adding pectinase into the pectin solution for enzymolysis; (5) after the enzymolysis is finished, separating by ultrafiltration; (6) concentrating the pectin solution into a concentrated solution by pressure distillation, and adding absolute ethyl alcohol for precipitation; (7) drying the pectin precipitate under normal pressure.
Description
Technical Field
The technology relates to the field of biological pharmacy, in particular to a preparation method of micromolecule citrus pectin.
Background
the citrus pectin is a natural polysaccharide high-molecular polymer extracted from citrus plants, the main chain is a long-chain structure formed by connecting D-galacturonic acid through α -1,4 glycosidic bonds, the side chain is composed of rhamnose, arabinose, galactose, xylose and the like, the pectin is a main component of dietary fiber of the seventh nutrient of a human body and has various edible and medicinal values, so the citrus pectin has wide application in the fields of food, medicine and the like, and small-molecule pectin (MCP) has high water solubility and can not be effectively added into health food because the pectin is not easily absorbed by intestinal tracts, enters blood circulation because of being well absorbed by the intestinal tracts, and has wider application in the fields of health food, medicine and the like.
MCP is a ligand of Galectin-3 (Galectin-3) and has a very strong affinity, and thus can be used as a competitive inhibitor of Galectin-3 ligand in vivo. When the concentration of MCP reaches a certain level in vivo, Galectin-3 in cancer cells reacts only with MCP and no longer binds to its ligand, thereby inhibiting the functional expression of Galectin-3 and preventing the formation of tumors. For established tumors, MCP can also prevent tumor cells from gaining nutrients from blood vessels, inhibiting their growth and metastasis by completely blocking the Galectin-3 site on the surface of tumor cells. Meanwhile, MCP also has the function of inhibiting homotypic aggregation of tumor cells, and can inhibit tumor angiogenesis and adhesion of tumor cells and endothelial cells. MCP has effects on all tumors with Galectin-3, and its efficiency is proportional to the expression of Galectin-3. MCP has been reported to be effective against 19 common tumors, including: prostate cancer, renal cancer, kaposi's sarcoma, chronic leukemia, breast cancer, sarcoma, ovarian cancer, colorectal cancer, throat cancer, lymphoma, melanoma, small intestine tumor, bladder tumor, lung cancer, bronchial cancer, pharyngeal squamous cell carcinoma, intestinal cancer, gastric cancer, skin cancer, liver cancer, brain cancer, glioma, thyroid cancer, and bone marrow cancer. MCP also has various biological activities, can reduce the concentration of cholesterol in blood, inhibit the accumulation of oil in liver, improve the immunity of organism and the like, and can be combined with heavy metal in blood to promote the discharge of MCP out of the body. Because MCP has various biological activities, the MCP has great application prospects in the aspects of treatment, free radical resistance, immunity regulation, blood fat reduction and the like.
At present, commercial pectin is extracted from plants, the molecular weight of the pectin directly extracted is between 5 and 30 ten thousand, while the molecular weight of MCP is generally between 5,000 and 3 ten thousand, and the pectin is obtained by degrading macromolecular pectin. The enzymolysis method is a common biological macromolecule degradation method, but needs to optimize reaction conditions to obtain MCP with higher purity and single molecular weight distribution, so the invention adopts the enzyme method to degrade macromolecular pectin to produce MCP, and aims to provide the MCP preparation method which has simple operation, single molecular weight distribution and easy industrial production.
Disclosure of Invention
The invention aims to develop a preparation method of micromolecule citrus pectin, which is simple to operate, single in molecular weight distribution and easy for industrial production.
A preparation method of micromolecule citrus pectin is characterized by sequentially comprising the following steps:
(1) pectin dissolution
Weighing citrus pectin, and adding the citrus pectin into water under stirring to dissolve, wherein the mass concentration of the pectin is 1-12%;
(2) centrifugal separation of pectin solution
Centrifuging to remove insoluble impurities after pectin is completely dissolved, wherein the centrifugation speed is 5,000-10,000, the centrifugation time is 5-20min, and the centrifugation temperature is 20-40 ℃;
(3) microfiltration separation of pectin solution
After the pectin is completely dissolved, carrying out microfiltration separation, wherein the temperature is 20-80 ℃, the pH value of the solution is 5-9, and removing particles with the particle size of more than 0.45 mu m;
(4) enzymolysis of pectin solution
Adding pectinase into the microfiltered pectin solution for degradation, wherein the consumption of the pectinase is 0.02-2% of the dry weight of the pectin, the enzymolysis temperature is 25-45 ℃, the enzymolysis time is 0.5-10h, and the mass concentration of the pectin is 1-12%;
(5) ultra-filtration separation of pectin solution
Ultrafiltering and separating the pectin solution after enzymolysis, wherein the molecular weight cut-off of the ultrafiltration membrane is 5,000-50,000dalton, and the operating pressure is 0.05-0.2 MPa;
(6) ethanol precipitation
Concentrating the ultrafiltered filtrate by reduced pressure distillation to obtain concentrated gelatin solution with mass concentration of 5-25%, adding anhydrous ethanol for precipitation at ethanol volume concentration of 50-90%, and ethanol precipitation temperature of 2-30 deg.C for 1-24 hr;
(7) drying pectin
Drying the pectin precipitate to obtain the product.
In the preparation method of the micromolecule citrus pectin, the mass concentration of the pectin in the step (1) is 1-12%, the stirring speed is 500-1,500rpm, the temperature is 20-80 ℃, and the stirring time is 1-4 h.
Further, the preparation method of the micromolecule citrus pectin comprises the step (2) of centrifugally separating pectin solution, carrying out high-speed centrifugation after the pectin is completely dissolved to remove insoluble impurities, wherein the centrifugal rotation speed is 5,000-10,000, the centrifugal time is 5-20min, and the centrifugal temperature is 20-40 ℃;
further, the step (3) of the preparation method of the micromolecule citrus pectin is microfiltration separation of the pectin solution, the pectin solution is sequentially filtered by microfiltration membranes with the aperture of 0.8 and the aperture of 0.45 mu m, the filtering temperature is 20-80 ℃, the pH value of the solution is 2.5-9, and particles with the particle size of more than 0.45 mu m are removed.
Further, the preparation method of the micromolecule citrus pectin comprises the step (4) of carrying out enzymolysis on the pectin solution, adding pectinase into the microfiltered pectin solution for degradation, wherein the consumption of the pectinase is 0.05-2% of the dry weight of the pectin, the enzymolysis temperature is 25-45 ℃, the enzymolysis time is 0.5-5h, and the mass concentration of the pectin is 1-12%;
further, the preparation method of the micromolecule citrus pectin comprises the step (5) of carrying out ultrafiltration separation on the pectin solution, carrying out ultrafiltration separation on the pectin solution after enzymolysis, wherein the molecular weight cut-off of an ultrafiltration membrane is 5,000-50,000dalton, and the operating pressure is 0.05-0.2 MPa;
further, the preparation method of the micromolecule citrus pectin comprises the step (6) of ethanol precipitation, the ultrafiltration filtrate is subjected to reduced pressure distillation and concentration, the mass concentration of the concentrated pectin solution is 5-25%, then absolute ethanol is added for precipitation, the final volume concentration of the ethanol is 50-90%, the ethanol precipitation temperature is 2-30 ℃, and the ethanol precipitation time is 1-24 hours;
further, the step (7) of the preparation method of the micromolecule citrus pectin is drying the pectin at normal pressure, wherein the drying temperature is 30-80 ℃.
The technical scheme provided by the invention has the following technical advantages:
(1) the low molecular pectin produced by the technical scheme provided by the invention has controllable molecular weight and narrower molecular weight distribution.
(2) The technical scheme provided by the invention is simple to operate and easy for process amplification and industrial production.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not to be construed as limiting the invention in any way. Any modification made by anyone within the scope of the claims of the present invention is still within the scope of the claims of the present invention.
Example 1
The invention provides a preparation method of micromolecule citrus pectin, which sequentially comprises the following steps:
(1) pectin dissolution
Weighing 60g of citrus pectin, adding into 1L of water under stirring to dissolve, wherein the mass concentration of the pectin is 6.0%, the stirring speed is 1,500rpm, the temperature is 30 ℃, and the stirring time is 1 h.
(2) The pectin solution is subjected to centrifugal separation,
centrifuging completely dissolved pectin solution to remove insoluble impurities at 9,000 rotation speed for 6min at 25 deg.C
(3) Microfiltration separation of pectin solution
The pectin solution completely dissolved is sequentially filtered and separated by microfiltration membranes with the aperture of 0.8 and the diameter of 0.45 mu m, the microfiltration temperature is 60 ℃, and the pH value of the solution is 7.
(4) Pectin enzymolysis
Adding pectinase into the pectin solution for degradation, wherein the consumption of the pectinase is 0.1% of the dry weight of pectin, the enzymolysis temperature is 40 ℃, the enzymolysis time is 4h, and the substrate concentration is 6.0%.
(5) Ultra-filtration separation of pectin solution
Carrying out ultrafiltration separation on the pectin solution subjected to enzymolysis, wherein the molecular weight cut-off of an ultrafiltration membrane is 30,000dalton, and the operating pressure is 0.1 MPa;
(6) ethanol precipitation
Concentrating the ultrafiltration filtrate by reduced pressure distillation, wherein the mass concentration of the concentrated gelatin solution is 15%, adding absolute ethanol for precipitation, the final volume concentration of the ethanol is 50%, the ethanol precipitation temperature is 20 ℃, and the ethanol precipitation time is 16 h;
(7) drying pectin
Drying the pectin precipitate at normal pressure at 60 deg.C.
The molecular weight of the low molecular pectin obtained by the preparation process is 15,600dalton, and the yield is 53.9%.
Example 2
The invention provides a preparation method of micromolecule citrus pectin, which sequentially comprises the following steps:
(1) pectin dissolution
Weighing 80g of citrus pectin, adding into 1L of water under stirring to dissolve, wherein the mass concentration of the pectin is 8.0%, the stirring speed is 1,500rpm, the temperature is 30 ℃, and the stirring time is 2 h.
(2) The pectin solution is subjected to centrifugal separation,
centrifuging completely dissolved pectin solution at 25 deg.C for 5min at 7,000 rotation speed to remove insoluble impurities
(3) Microfiltration separation of pectin solution
The pectin solution completely dissolved is sequentially subjected to microfiltration membrane separation with the aperture of 0.8 and the aperture of 0.45 mu m, the microfiltration temperature is 70 ℃, and the pH value of the solution is 7.
(4) Pectin enzymolysis
Adding pectinase into the pectin solution for degradation, wherein the consumption of the pectinase is 0.15% of the dry weight of pectin, the enzymolysis temperature is 40 ℃, the enzymolysis time is 3h, and the substrate concentration is 8.0%.
(5) Ultra-filtration separation of pectin solution
Carrying out ultrafiltration separation on the pectin solution subjected to enzymolysis, wherein the molecular weight cut-off of an ultrafiltration membrane is 30,000dalton, and the operating pressure is 0.15 MPa;
(6) ethanol precipitation
Concentrating the ultrafiltration filtrate by reduced pressure distillation, wherein the mass concentration of the concentrated gelatin solution is 10%, then adding absolute ethanol for precipitation, the final volume concentration of the ethanol is 60%, the alcohol precipitation temperature is 20 ℃, and the alcohol precipitation time is 4 h;
(7) drying pectin
Drying the pectin precipitate at normal pressure at 60 deg.C.
The molecular weight of the low molecular pectin obtained by the preparation process is 14,700dalton, and the yield is 51.8%.
Example 3
The invention provides a preparation method of micromolecule citrus pectin, which sequentially comprises the following steps:
(1) pectin dissolution
Weighing 4.0g of citrus pectin, adding into 1L of deionized water under stirring to dissolve, wherein the mass concentration of the pectin is 4.0%, the stirring speed is 1,500rpm, the temperature is 30 ℃, and the stirring time is 3 h.
(2) The pectin solution is subjected to centrifugal separation,
centrifuging completely dissolved pectin solution at 25 deg.C for 6min at 7,000 rotation speed to remove insoluble impurities
(3) Microfiltration separation of pectin solution
The pectin solution completely dissolved is sequentially filtered and separated by microfiltration membranes with the aperture of 0.8 and the aperture of 0.45 mu m, the microfiltration temperature is 70 ℃, and the pH value of the solution is 8.
(4) Pectin enzymolysis
Adding pectinase into the pectin solution for degradation, wherein the consumption of the pectinase is 0.08% of the dry weight of pectin, the enzymolysis temperature is 40 ℃, the enzymolysis time is 2h, and the substrate concentration is 4.0%.
(5) Ultra-filtration separation of pectin solution
Carrying out ultrafiltration separation on the pectin solution subjected to enzymolysis, wherein the molecular weight cut-off of an ultrafiltration membrane is 30,000dalton, and the operating pressure is 0.1 MPa;
(6) ethanol precipitation
Concentrating the ultrafiltration filtrate by reduced pressure distillation, wherein the mass concentration of the concentrated gelatin solution is 20%, adding anhydrous ethanol for precipitation, the final volume concentration of the ethanol is 70%, the ethanol precipitation temperature is 20 ℃, and the ethanol precipitation time is 4 h;
(7) drying pectin
Drying the pectin precipitate at normal pressure at 50 deg.C.
The molecular weight of the low molecular pectin obtained by the preparation process is 13,200dalton, and the yield is 48.7%.
Claims (5)
1. A preparation method of micromolecule citrus pectin is characterized by sequentially comprising the following steps:
(1) pectin dissolution
Weighing citrus pectin, and adding the citrus pectin into water under stirring to dissolve, wherein the mass concentration of the pectin is 1-12%;
(2) centrifugal separation of pectin solution
Centrifuging to remove insoluble impurities after pectin is completely dissolved, wherein the centrifugation speed is 5000-10000, the centrifugation time is 5-20min, and the centrifugation temperature is 20-40 ℃;
(3) microfiltration separation of pectin solution
After the pectin is completely dissolved, carrying out microfiltration separation, wherein the temperature is 20-80 ℃, the pH value of the solution is 5-9, and removing particles with the particle size of more than 0.45 mu m;
(4) enzymolysis of pectin solution
Adding pectinase into the microfiltered pectin solution for degradation, wherein the consumption of the pectinase is 0.05-2% of the dry weight of the pectin, the enzymolysis temperature is 25-45 ℃, the enzymolysis time is 0.5-10h, and the mass concentration of the pectin is 1-12%;
(5) ultra-filtration separation of pectin solution
Carrying out ultrafiltration separation on the pectin solution subjected to enzymolysis, wherein the cut-off molecular weight of an ultrafiltration membrane is 5000-50000dalton, and the operating pressure is 0.05-0.2 MPa;
(6) ethanol precipitation
Concentrating the ultrafiltered filtrate by reduced pressure distillation to obtain concentrated gelatin solution with mass concentration of 5-25%, adding anhydrous ethanol for precipitation at alcohol precipitation temperature of 2-30 deg.C and final volume concentration of 50-90%, and precipitating for 1-24 hr;
(7) drying pectin
Drying the pectin precipitate to obtain the product.
2. The method for producing low-molecular pectin according to claim 1, wherein: in the dissolving process in the step (1), the stirring speed is 500-1,500rpm, the temperature is 20-80 ℃, and the stirring time is 1-4 h.
3. The method for producing low-molecular pectin according to claim 1, wherein: and (3) the microfiltration of the pectin solution in the step (2) is sequentially filtered by microfiltration membranes with 2 kinds of pore sizes, wherein the pore sizes are 0.8 mu m and 0.45 mu m.
4. The method for producing low-molecular pectin according to claim 1, wherein: and (4) drying the pectin precipitate at normal pressure in the step (7), wherein the drying temperature is 30-80 ℃.
5. A low molecular pectin obtainable by the process according to any one of claims 1 to 4, wherein: the molecular weight of the low molecular pectin ranges from 5,000 to 3 ten thousand.
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Cited By (3)
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| CN111840650A (en) * | 2020-07-28 | 2020-10-30 | 中国医学科学院生物医学工程研究所 | Collagen-based modified citrus pectin composite material and preparation method and application thereof |
| CN113462731A (en) * | 2021-06-29 | 2021-10-01 | 北京化工大学 | Preparation method of micromolecule pectin |
| CN113480676A (en) * | 2021-09-08 | 2021-10-08 | 天津银杉科技有限公司 | Oligogalacturonic acid polysaccharide, compound, preparation method and application thereof |
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| CN113480676A (en) * | 2021-09-08 | 2021-10-08 | 天津银杉科技有限公司 | Oligogalacturonic acid polysaccharide, compound, preparation method and application thereof |
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